Defining early steps in mRNA transport: mutant mRNA in myotonic dystrophy type I is blocked at entry into SC-35 domains

In myotonic dystrophy type 1 (DM1), triplet repeat expansion in the 3′ untranslated region of dystrophia myotonica protein kinase (DMPK) causes the nuclear retention of mutant messenger RNA (mRNA). Although the DMPK gene locus positions precisely at the outer edge of a factor-rich SC-35 domain, the normal mRNA consistently accumulates within the domain, and this RNA is depleted upon transcriptional inhibition. In DM1, mutant transcripts detach from the gene but accumulate in granules that abut but do not enter SC-35 domains, suggesting that RNA entry into the domain is blocked. Despite their exclusion from these compartments, mutant transcripts are spliced. MBNL1 (muscleblind-like protein 1) is an alternative splicing factor that becomes highly concentrated with mutant RNA foci. Small interfering RNA–mediated knockdown of MBNL1 promotes the accumulation or entry of newly synthesized mutant transcripts in the SC-35 domain. Collectively, these data suggest that an initial step in the intranuclear path of some mRNAs is passage from the gene into an SC-35 domain and implicate these structures in postsplicing steps before export

Clustering of multiple specific genes and gene-rich R-bands around SC-35 domains: evidence for local euchromatic neighborhoods

Typically, eukaryotic nuclei contain 10–30 prominent domains (referred to here as SC-35 domains) that are concentrated in mRNA metabolic factors. Here, we show that multiple specific genes cluster around a common SC-35 domain, which contains multiple mRNAs. Nonsyntenic genes are capable of associating with a common domain, but domain “choice” appears random, even for two coordinately expressed genes. Active genes widely separated on different chromosome arms associate with the same domain frequently, assorting randomly into the 3–4 subregions of the chromosome periphery that contact a domain. Most importantly, visualization of six individual chromosome bands showed that large genomic segments (∼5 Mb) have striking differences in organization relative to domains. Certain bands showed extensive contact, often aligning with or encircling an SC-35 domain, whereas others did not. All three gene-rich reverse bands showed this more than the gene-poor Giemsa dark bands, and morphometric analyses demonstrated statistically significant differences. Similarly, late-replicating DNA generally avoids SC-35 domains. These findings suggest a functional rationale for gene clustering in chromosomal bands, which relates to nuclear clustering of genes with SC-35 domains. Rather than random reservoirs of splicing factors, or factors accumulated on an individual highly active gene, we propose a model of SC-35 domains as functional centers for a multitude of clustered genes, forming local euchromatic “neighborhoods.

Regulation of X-linked gene expression during early mouse development by

Mammalian X-linked gene expression is highly regulated as female cells contain two and male one X chromosome (X). To adjust the X gene dosage between genders, female mouse preimplantation embryos undergo an imprinted form of X chromosome inactivation (iXCI) that requires both Rlim (also known as Rnf12) and the long non-coding RNA Xist. Moreover, it is thought that gene expression from the single active X is upregulated to correct for bi-allelic autosomal (A) gene expression. We have combined mouse genetics with RNA-seq on single mouse embryos to investigate functions of Rlim on the temporal regulation of iXCI and Xist. Our results reveal crucial roles of Rlim for the maintenance of high Xist RNA levels, Xist clouds and X-silencing in female embryos at blastocyst stages, while initial Xist expression appears Rlim-independent. We find further that X/A upregulation is initiated in early male and female preimplantation embryos

Rlim-Dependent and -Independent Pathways for X Chromosome Inactivation in Female ESCs

During female mouse embryogenesis, two forms of X chromosome inactivation (XCI) ensure dosage compensation from sex chromosomes. Beginning at the four-cell stage, imprinted XCI (iXCI) exclusively silences the paternal X (Xp), and this pattern is maintained in extraembryonic cell types. Epiblast cells, which give rise to the embryo proper, reactivate the Xp (XCR) and undergo a random form of XCI (rXCI) around implantation. Both iXCI and rXCI depend on the long non-coding RNA Xist. The ubiquitin ligase RLIM is required for iXCI in vivo and occupies a central role in current models of rXCI. Here, we demonstrate the existence of Rlim-dependent and Rlim-independent pathways for rXCI in differentiating female ESCs. Upon uncoupling these pathways, we find more efficient Rlim-independent XCI in ESCs cultured under physiological oxygen conditions. Our results revise current models of rXCI and suggest that caution must be taken when comparing XCI studies in ESCs and mice

Translating dosage compensation to trisomy 21

Down syndrome is the leading genetic cause of intellectual disabilities, occurring in 1 out of 700 live births. Given that Down syndrome is caused by an extra copy of chromosome 21 that involves over-expression of 400 genes across a whole chromosome, it precludes any possibility of a genetic therapy. Our lab has long studied the natural dosage compensation mechanism for X chromosome inactivation. To “dosage compensate” X-linked genes between females and males, the X-linked XIST gene produces a large non-coding RNA that silences one of the two X chromosomes in female cells. The initial motivation of this study was to translate the natural mechanisms of X chromosome inactivation into chromosome therapy for Down syndrome. Using genome editing with zinc finger nucleases, we have successfully inserted a large XIST transgene into Chromosome 21 in Down syndrome iPS cells, which results in chromosome-wide transcriptional silencing of the extra Chromosome 21. Remarkably, deficits in proliferation and neural growth are rapidly reversed upon silencing one chromosome 21. Successful trisomy silencing in vitro surmounts the major first step towards potential development of “chromosome therapy” for Down syndrome. The human iPSC-based trisomy correction system we established opens a unique opportunity to identify therapeutic targets and study transplantation therapies for Down syndrome

AURKB-mediated effects on chromatin regulate binding versus release of XIST RNA to the inactive chromosome

How XIST RNA strictly localizes across the inactive X chromosome is unknown; however, prophase release of human XIST RNA provides a clue. Tests of inhibitors that mimic mitotic chromatin modifications implicated an indirect role of PP1 (protein phosphatase 1), potentially via its interphase repression of Aurora B kinase (AURKB), which phosphorylates H3 and chromosomal proteins at prophase. RNA interference to AURKB causes mitotic retention of XIST RNA, unlike other mitotic or broad kinase inhibitors. Thus, AURKB plays an unexpected role in regulating RNA binding to heterochromatin, independent of mechanics of mitosis. H3 phosphorylation (H3ph) was shown to precede XIST RNA release, whereas results exclude H1ph involvement. Of numerous Xi chromatin (chromosomal protein) hallmarks, ubiquitination closely follows XIST RNA retention or release. Surprisingly, H3S10ph staining (but not H3S28ph) is excluded from Xi and is potentially linked to ubiquitination. Results suggest a model of multiple distinct anchor points for XIST RNA. This study advances understanding of RNA chromosome binding and the roles of AURKB and demonstrates a novel approach to manipulate and study XIST RNA

A multifaceted FISH approach to study endogenous RNAs and DNAs in native nuclear and cell structures

Fluorescence in situ hybridization (FISH) is not a singular technique, but a battery of powerful and versatile tools for examining the distribution of endogenous genes and RNAs in precise context with each other and in relation to specific proteins or cell structures. This unit offers the details of highly sensitive and successful protocols that were initially developed largely in our lab and honed over a number of years. Our emphasis is on analysis of nuclear RNAs and DNA to address specific biological questions about nuclear structure, pre-mRNA metabolism, or the role of noncoding RNAs; however, cytoplasmic RNA detection is also discussed. Multifaceted molecular cytological approaches bring precise resolution and sensitive multicolor detection to illuminate the organization and functional roles of endogenous genes and their RNAs within the native structure of fixed cells. Solutions to several common technical pitfalls are discussed, as are cautions regarding the judicious use of digital imaging and the rigors of analyzing and interpreting complex molecular cytological results

Ubiquitinated proteins including uH2A on the human and mouse inactive X chromosome: enrichment in gene rich bands

The inactive X chromosome (Xi) forms a heterochromatic structure in the nucleus that is known to have several modifications to specific histones involving acetylation or methylation. Using three different antibodies in four different cell lines, we demonstrate that the Xi in human and mouse cells is highly enriched in ubiquitinated protein(s), much of which is polyubiquitinated. This ubiquitination appears specific for the Xi as it was not observed for centromeres or other regions of heterochromatin. Results using an antibody specific to ubiquitinated H2A provide a clear link between H2A ubiquitination and gene repression, as visualized across an entire inactive chromosome. Interestingly, the ubiquitination of the chromosome persists into mitosis and can be seen in a reproducible banded pattern. This pattern matches that of Xist RNA which forms bands as it detaches from the mitotic X chromosome. Both ubiquitination and Xist RNA appear enriched in gene dense regions and depleted in gene poor bands, but do not correlate with L1 LINE elements which have been suggested as key to X-inactivation. These results provide evidence that ubiquitination along with Xist RNA plays an important role in the formation of facultative heterochromatin during X-inactivation

Expression of the repeat genome and aberrant epigenetic factors in cancer

Nearly half of the human genome consists of noncoding repetitive DNA elements, including tandem satellite repeats in large blocks at the pericentric regions of chromosomes and intergenic repetitive elements. While both repeat types were long thought to remain mostly silent, recent evidence indicates that repeats can be expressed, but the extent and regulation of their expression or their potential function(s) remain to be elucidated. Due to their critical location within regions vital for cell division, it is expected that tight regulation of pericentric satellite sequences is essential for both epigenetic and genetic stability. Our data suggests aberrant expression of pericentric satellite RNA is tightly linked to epigenetic misregulation in cancer. It is well known that epigenetic changes can be important in cancer initiation and progression, but studies have focused primarily on the inappropriate silencing and methylation of tumor suppressor genes. While pathologists have long noted the loss of heterochromatic organization in cancer nuclei, and hypomethylation of satellite DNA has been observed, the misregulation of repeat RNAs has only recently been described. Our results provide a link between overexpression of repeat RNAs and aberrant distribution of epigenetic factors in cancer. Our data suggests regulation of the repeat genome has potentially important roles in both normal and neoplastic cells in their ability to affect distribution and recruitment of epigenetic factors

Molecular anatomy of a speckle

Direct localization of specific genes, RNAs, and proteins has allowed the dissection of individual nuclear speckles in relation to the molecular biology of gene expression. Nuclear speckles (aka SC35 domains) are essentially ubiquitous structures enriched for most pre-mRNA metabolic factors, yet their relationship to gene expression has been poorly understood. Analyses of specific genes and their spliced or mature mRNA strongly support that SC35 domains are hubs of activity, not stores of inert factors detached from gene expression. We propose that SC35 domains are hubs that spatially link expression of specific pre-mRNAs to rapid recycling of copious RNA metabolic complexes, thereby facilitating expression of many highly active genes. In addition to increasing the efficiency of each step, sequential steps in gene expression are structurally integrated at each SC35 domain, consistent with other evidence that the biochemical machineries for transcription, splicing, and mRNA export are coupled. Transcription and splicing are subcompartmentalized at the periphery, with largely spliced mRNA entering the domain prior to export. In addition, new findings presented here begin to illuminate the structural underpinnings of a speckle by defining specific perturbations of phosphorylation that promote disassembly or assembly of an SC35 domain in relation to other components. Results thus far are consistent with the SC35 spliceosome assembly factor as an integral structural component. Conditions that disperse SC35 also disperse poly(A) RNA, whereas the splicing factor ASF/SF2 can be dispersed under conditions in which SC35 or SRm300 remain as intact components of a core domain